Blood biomarker that predicts persistent cognitive dysfunction after concussion

ABSTRACT

The invention relates to methods for providing prognosis, diagnosis, and treatment of a mild traumatic brain injury (mTBI) in a computed tomography (CT)-negative subject. The invention further relates to monitoring the severity of brain damage resulting from TBI in a subject and determining the prognosis of a subject that has suffered from mTBI. This invention also relates to methods of predicting who is at risk for developing brain damage and long-term dysfunction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase application of PCT InternationalApplication No. PCT/US14/27716, International filing date Mar. 14, 2013,which claims priority to and the benefit of U.S. Patent Application61/792,420, filed Mar. 15, 2013, all of which are hereby incorporated byreference herein in their entirety.

GOVERNMENT INTEREST

This invention was made with government support under grant numberNS056202 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF INVENTION

The invention relates to methods for providing prognosis, diagnosis, andtreatment for a mild traumatic brain injury (mTBI) in a computedtomography (CT)-negative subject. The invention further relates tomonitoring the severity of brain damage resulting from mTBI in a subjectand determining the prognosis of a subject that has suffered from mTBI.This invention also relates to methods of predicting who is at risk fordeveloping brain damage and long-term dysfunction.

BACKGROUND OF THE INVENTION

Mild traumatic brain injury (mTBI), alternatively referred to asconcussion, is the most common neurological injury and affects over 1.5million children and adults each year in the United States alone, andhundreds of thousands of military personnel worldwide. mTBI is typicallyundetectable with computed tomography (CT), yet can elicit long-term andclinically significant brain dysfunction in approximately 15-30% ofcases. Histopathological and biomechanical findings in experimentalanimal models and human cases that have come to autopsy suggest that themain underlying structural correlate for long-term functional impairmentafter mTBI is diffuse axonal injury (DAI), resulting from headrotational acceleration at the moment of injury. Developingneuroradiological methods such as diffusion tensor imaging (DTI) haveshown promise for the detection of white matter structural abnormalitiesafter mTBI, but collectively these studies have yielded inconsistentresults. Consequently, new approaches are urgently needed for the rapididentification of mTBI patients at risk of developing brain damage andpersistent disability.

Blood-based biomarkers for brain damage have long been evaluated aspotential prognostic measures in mTBI, but none has emerged thus far asa means of identifying at an early and potentially treatable stage thosecases of mTBI with evolving brain damage leading to long-termdysfunction. For example, a number of proteins expressed predominantlyin the nervous system become detectable in the blood during the acutepost-injury period in some mTBI cases, including the astrocyte-enrichedproteins S100β and glial fibrillary acidic protein (GFAP), along withthe neuron-enriched neuron-specific enolase (NSE), ubiquitin C-terminalhydrolase L1 (UCH-L1), and a proteolytic fragment of tau.

Blood levels of these markers for brain damage are reportedly elevatedfollowing injuries categorized as mild based on clinical examinationsusing the Glasgow Coma Scale. However, these studies have focusedpredominantly on TBI cases that also show head CT abnormalities, andbased on the positive CT findings these patients would be diagnosed withmoderate TBI or “complicated” mTBI at most centers. Positive CT findingsare known to be associated with poorer long-term outcomes after TBI, andthe presence of intracranial hemorrhages suggests that the blood-brainbarrier exhibits at least transient permeability that could impactblood-based biomarker measures. Unfortunately, for the much more commoninstances of CT-negative mTBI, blood-based markers for brain injury haveyet to be discovered that are strong predictors of structural damage andlong-term functional outcome.

Therefore, there is a need in the art for neurodegeneration biomarkersreleased from degenerating neurons that are indicative of CT-negativemTBI. The present invention addresses this need by providing a methodfor using calpain-cleaved αII-spectrin N-terminal fragment (SNTF) as amechanism-based marker for the calpain-associated necrotic mode ofneurodegeneration following mTBI.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a method of providing aprognosis for a subject having suffered from a computed tomography(CT)-negative mild traumatic brain injury (mTBI), said methodcomprising: obtaining a biological sample from said subject; analyzingsaid sample to determine the level of a biomarker associated with thepresence of mTBI, and comparing said level of said biomarker to thelevel of a pre-determined standard, wherein said biomarker is acalpain-cleaved αII-spectrin N-terminal fragment (SNTF), therebyproviding a prognosis for subject having suffered from mTBI.

In another embodiment, the invention relates to a method of monitoringresponse to therapy in a subject having suffered from a computedtomography (CT)-negative mild traumatic brain injury (mTBI), said methodcomprising: obtaining a biological sample from said subject receivingsaid therapy; analyzing said sample to determine the level of abiomarker associated with the presence of mTBI, and comparing said levelof said biomarker to the level of a pre-determined standard, therebymonitoring a response to therapy in a subject having suffered from mTBI.

In another embodiment, the invention relates to a method for identifyinga subject at risk of suffering from a mild traumatic brain injury(mTBI)-associated abnormality in white matter structure or a long-termdysfunction, said method comprising: obtaining a biological sample fromsaid subject; analyzing said sample to determine the level of abiomarker associated with the presence of mTBI, and comparing said levelof said biomarker to the level of a pre-determined standard, whereinsaid biomarker is a calpain-cleaved αII-spectrin N-terminal fragment(SNTF), thereby identifying a subject at risk of suffering from a mildtraumatic brain injury (mTBI)-associated abnormality in white matterstructure or a long-term dysfunction.

In another embodiment, the invention relates to a method for diagnosinga mild traumatic brain injury (mTBI) in a subject having a negativecomputed tomography (CT) test result, said method comprising: obtaininga biological sample from said subject; analyzing said sample todetermine the level of a biomarker associated with the presence of saidmTBI, and comparing said level of said biomarker to the level of apre-determined standard, thereby diagnosing mTBI.

In another embodiment, the invention relates to a method for treating amild traumatic brain injury (mTBI) in a subject having a negativecomputed tomography (CT) test result, said method comprising: obtaininga biological sample from said subject; analyzing said sample todetermine the level of a biomarker associated with the presence of saidmTBI, comparing said level of said biomarker to the level of apre-determined standard, thereby prognosing or diagnosing mTBI, andtreating said subject based on the prognosis or diagnosis of said mTBI.

Other features and advantages of the present invention will becomeapparent from the following detailed description examples and figures.It should be understood, however, that the detailed description and thespecific examples while indicating preferred embodiments of theinvention are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Plasma SNTF discriminates 3 months changes in cognitiveperformance after mTBI. The difference in SDMTO scores between the acute(1-4 day) and chronic (3 months) post-injury periods is plotted as afunction of dichotomized plasma SNTF levels on the day of mTBI. Thedifference in cognitive performance recoveries between the biomarkernegative and positive Mtbi groups is significant (p<0.03, two-tailedt-test).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, provided herein is a method for providing prognosis,diagnosis, or treatment for a mild traumatic brain injury (mTBI) in asubject having a negative computed tomography (CT) test result, saidmethod comprising: obtaining a biological sample from said subject;analyzing said sample to determine the level of a biomarker associatedwith the presence of said mTBI, and comparing said level of saidbiomarker to the level of a pre-determined standard, thereby providingprognosis, diagnosis, or treatment for said mTBI. In one embodiment, thebiomarker obtained from the biological sample according to the methodsprovided herein is correlated to brain damage and long-term functionaldisability.

In one embodiment, the standard is determined by measuring the level ofsaid biomarker in a subject or pool of subjects having sustained anorthopedic injury or in a normal uninjured subject or pool of subjects.In another embodiment, the standard is taken from a subject or pool ofsubjects correctly diagnosed as being normal or healthy.

In one embodiment, diagnosing a mTBI further permits assesing a risk ofsaid mTBI evolving to brain damage and leading to long-term dysfunction.In another embodiment, assessment of a risk of said mTBI evolving tolong-term dysfunction permits therapeutic intervention at an earlystage.

In one embodiment, the type of brain damage associated with mTBI is awhite matter structural abnormality. In another embodiment, the whitematter structural abnormality or damage is in the corpus callosumregion. In another embodiment, the abnormality or damage is in theuncinate fasciculus. In another embodiment, the abnormality or damage isin the right brain frontal lobe. In another embodiment, the abnormalityor damage is in the left frontal lobe. In another embodiment, theabnormality or damage is diffuse axonal injury (DAI).

Brain damage can be assessed by neuroimaging techniques and long-termcongnitive assessment. In one embodiment, the present invention providesfor a combined assessment of brain damage comprising assessing abiomarker level (or panel of biomarkers) in a biological sample obtainedfrom a subject having suffered a mTBI, by using diffusion tensor imaging(DTI), and by using neuropsychological/behavioral methods. Diffusiontensor imaging (DTI) is well known in the art and it is used to measurethe fractional anisotropy (FA) and the apparent diffusion coefficient(ADC) in a biological tissue.

In one embodiment, a concussion is a mTBI. In another embodiment, mTBIis caused by a head injury, where the head injury is, in anotherembodiment, blunt trauma, acceleration, or deceleration forces. It willbe appreciated that such head injuries can be characterized by havingone or more of the following conditions: (1) observed or self-reportedcontusion, disorientation, or impaired consciousness, dysfunction ofmemory at the time of the injury, loss of consciousness lasting lessthan 30 minutes; and, (2) symptoms such as headache, dizziness, fatigue,irritability, and poor concentration soon after the injury. Headinjuries are also categorized as mild based on clinical examinationsusing the Glasgow Coma Scale. In one embodiment, the head injury has aGlasgow Coma Scale score of 13-15 upon examination at an emergencycenter, with no abnormal findings on head CT, duration of loss ofconsciousness for no more than 30 minutes, post-traumatic amnesia forless than 24 hours, and an Abbreviated Injury Score (AIS) S3 and an ISSof <12 modified to exclude the head region.

In one embodiment, the level of the biomarker in a biological sampleobtained from a subject, as provided herein, is independently associatedwith mTBI and clinically important parameters in mTBI. In anotherembodiment, the biomarker is indicative of the severity of saidsubject's condition. In another embodiment, comparing said level of saidbiomarker in said biological sample to said level of said standardpermits diagnosing the severity of mTBI. In another embodiment,sustained levels of said biomarker are associated with a subsequentincreased risk of long-term neurological dysfunction.

In one embodiment, a biological sample is blood, sera, plasma, cerebrospinal fluid (CNS), DNA, tissue biopsy, organ biopsy or theircombination.

In one aspect, provided herein is a method of monitoring response totherapy in a subject having suffered from a computed tomography(CT)-negative mild traumatic brain injury (mTBI), said methodcomprising: obtaining a biological sample from said subject receivingsaid therapy; analyzing said sample to determine the level of abiomarker associated with the presence of mTBI, and comparing said levelof said biomarker to the level of a pre-determined standard, therebymonitoring a response to therapy in a subject having suffered from mTBI.

In one embodiment, the biomarker in said biological sample is correlatedto brain damage and long-term functional disability. In anotherembodiment, monitoring response to a therapy further permits optimallyadjusting said therapy to reduce a risk of said mTBI evolving to braindamage leading to long-term dysfunction. In another embodiment,comparing a level of the biomarker or a panel of biomarkers in saidbiological sample to said level of said standard or a pool of standardspermits monitoring the response of said therapy. In another embodiment,wherein decreasing levels of said biomarker as a result of said therapyare associated with a subsequent decreased risk of long-termneurological dysfunction.

In one aspect, provided herein is a method of providing a prognosis fora subject having suffered from a computed tomography (CT)-negative mildtramatic brain injury (mTBI), said method comprising: obtaining abiological sample from said subject; analyzing said sample to determinethe level of a biomarker associated with the presence of mTBI, andcomparing said level of said biomarker to the level of a pre-determinedstandard, wherein said biomarker is calpain-cleaved αII-spectrinN-terminal fragment (SNTF), thereby providing a prognosis for subjecthaving suffered from mTBI.

In one aspect, provided herein is a method for identifying a subject atrisk of suffering from a mild traumatic brain injury (mTBI)-associatedabnormality in white matter structure or a long-term dysfunction, saidmethod comprising: obtaining a biological sample from said subject;analyzing said sample to determine the level of a biomarker associatedwith the presence of mTBI, and comparing said level of said biomarker tothe level of a pre-determined standard, wherein said biomarker is acalpain-cleaved αII-spectrin N-terminal fragment (SNTF), therebyidentifying a subject at risk of suffering from a mild traumatic braininjury (mTBI)-associated abnormality in white matter structure or along-term dysfunction.

In one embodiment, the standard is determined by measuring the level ofsaid biomarker or a pool of biomarkers in a subject having sustained anorthopedic injury or in a normal uninjured subject. In anotherembodiment, wherein comparing the level of biomarker or pool ofbiomarkers in said biological sample to said level of said standard orpool of standards permits identifying a subject at risk of sufferingfrom a mild traumatic brain injury (mTBI)-associated abnormality inwhite matter structure or a long-term dysfunction.

In one embodiment, the biomarker provided herein is a calpain-cleavedαII-spectrin N-terminal fragment (SNTF). In one embodiment, SNTF is amarker for mTBI and its blood levels are related to white matterabnormalities and long-term functional disability. In anotherembodiment, detecting the presence of SNTF in a biological sampleobtained from a subject shortly after mTBI is indicative of a risk ofdeveloping white matter tract structural damage and long-termdisability.

The present invention demonstrates that the blood level of theneurodegeneration biomarker SNTF identifies patients with mTBI likely tohave both white matter changes with advanced neuroimaging suggestive ofDAI, and also cognitive dysfunction that persists for at least 3 months(see Examples herein).

In some embodiments, the injury-induced elevation in plasma SNTF in mTBIcases triggers calpain activation and spectrin degradation withinvulnerable axons, followed by efflux of the protein fragment into thebrain parenchyma and bloodstream in association with the axon tractdamage underlying brain functional impairment.

In one embodiment, detecting the presence of neurodegeneration markersin a biological sample obtained from a subject shortly after mTBI isindicative of a risk of developing white matter tract structural damageand long-term disability. In another embodiment, detecting the presenceof neurodegeneration markers in a biological sample obtained from aCT-negative subject shortly after mTBI is indicative of a risk ofdeveloping white matter tract structural damage and long-termdisability.

In one embodiment the invention further encompasses functional variantsof the SNTF. In another embodiment, the biomarker is the N-terminalfragment of α-spectrin. α-spectrin (alpha chain of spectrin) is aprotein that in humans is encoded by the SPTA1 gene. Spectrin is anactin crosslinking and molecular scaffold protein that links the plasmamembrane to the actin cytoskeleton, and functions in the determinationof cell shape, arrangement of transmembrane proteins, and organizationof organelles. It is a tetramer made up of alpha-beta dimers linked in ahead-to-head arrangement. This gene is one member of a family ofalpha-spectrin genes. The encoded protein is primarily composed of 22spectrin repeats which are involved in dimer formation. It forms weakertetramer interactions than non-erythrocytic alpha spectrin, which mayincrease the plasma membrane elasticity and deformability of red bloodcells. Mutations in this gene result in a variety of hereditary redblood cell disorders, including elliptocytosis type 2,pyropoikilocytosis, and spherocytic hemolytic anemia.

In one embodiment, the level of the biomarker provided herein iselevated in a biological sample obtained from a patient having sufferedmTBI. In another embodiment, the biomarker is expressed in a biologicalsample obtained from a subject having suffered from mTBI.

In another embodiment, provided herein are methods for analyzing nucleicacid expression of the biomarkers provided herein. It will beappreciated that the term “nucleic acid” can encompass phosphate esterpolymeric form of ribonucleosides (adenosine, guanosine, uridine orcytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine,deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”).

In another embodiment, the invention encompasses use of SNTF as a singleor panel of biomarkers for potential clinical diagnosis, risk assessmentor prognosis. In another embodiment, this would lead to improved riskstratification and the use of directed therapy to modify known factorsthat are associated with worse outcomes.

In another embodiment, the methods provided herein further comprisejointly using first a SNTF biomarker and an additional neurodegenerativebiomarker. In another embodiment, the association of SNFT with adverseoutcomes is independent of the additional biomarker. In anotherembodiment, the assessment of the biomarkers jointly improves riskassessment over either marker alone in subjects with mTBI.

In another embodiment, mTBI leads to acute brain damage and long-termdysfunction. In another embodiment, the long-term dysfunction is asensory disfuction. In another embodiment, the dysfunction is a motordysfunction.

In one embodiment, the subject is a human subject. In anotherembodiment, the subject is being monitored for brain damage. In anotherembodiment, the subject is undergoing therapy for brain damage.

The term “standard” encompasses pooled samples from healthy subjects. Inanother embodiment, the standard may be ethnically- or gender- orage-matched recipients. It is to be understood that the standard may bederived from any subject, or pool of subjects, whose biomarker levelprofile is sufficient to detect even minute relative differences inbiomarker levels, when compared to a test sample, or in anotherembodiment, to a subject that has mTBI with evolving brain damageleading to long-term dysfunction. In another embodiment, a standard isdetermined as such by a skilled artisan.

In another embodiment, the standard is the average biomarker level of atleast one biomarker in a biological sample of the invention taken from apool of subjects. In another embodiment, the standard is the meanbiomarker level profile taken from a pool of subjects.

In another embodiment, the standard is the median biomarker level in abiological sample taken from a pool of subjects. In another embodiment,the standard is the median biomarker level of one biomarker of theinvention taken from a pool of subjects. In another embodiment, thestandard is the median biomarker level of at least one or morebiomarkers of the invention taken from a pool of subjects.

In another embodiment, the method involves the detection of level ofsaid biomarker in a biological sample. In another embodiment, the levelin of the biomarker in a biological sample obtained from a subject asprovided herein, is indicative of the severity of the subject'scondition. In yet another embodiment, the biomarker level is elevatedrelative to the level of a standard. In yet another embodiment,comparing the the biomarker level in a biological sample of a standardpermitsmeasuring the severity of brain damage in a subject. In anotherembodiment, it permits diagnosing the severity of brain damage in thesubject. In another embodiment, it permits monitoring the severity braindamage in the subject. In another embodiment, it permitsdetermining theprognosis of brain damage in the subject. In another embodiment, itpermits monitoring the therapeutic response of a subject following mTBI.In another embodiment, it permits monitoring said subject for long-termdysfunction.

In another embodiment the biomarker is present in blood, sera, plasma,saliva, sperm, urine, mucous, cerebral spinal fluid, or any combinationthereof and such presence is independently associated mTBI. In anotherembodiment a biomarker is not present or present in negligible levels inblood, sera, plasma, saliva, sperm, urine, mucous, cerebral spinalfluid, or any combination thereof of a normal subject.

In one embodiment, the level of the biomarker is determined by methodsknown in the art and include, but are not limited to, PCR, Microarrayassays, Immunoblots, notherns, ELISA, fluorescence-based methods(Immunofluorescence, FACS), mass spectrometry, and the like. In anotherembodiment, any other method known in the art is used formeasuring/analyzing/quantifying the level of a biomarker providedherein.

As used herein, the term “expression” refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from anucleic acid fragment or fragments of the invention. Expression alsorefers to the translation of mRNA into a polypeptide.

In another embodiment the biomarker/biomarkers expression is/aretissue-specific. In another embodiment, the biomarker/biomarkersexpression is/are global. In another embodiment, thebiomarker/biomarkers is/are expression is systemic.

In one embodiment, the present invention encompasses several examples ofa biological sample. In another embodiment, the biological sample iscells, or in another embodiment tissue or in another embodimentperipheral blood. In one embodiment, the biological sample is obtainedfrom a normal subject. The biological sample may, in one embodiment, bea sample of body fluid. In another embodiment, biological samples referto, blood, serum, plasma, sperm, urine, mucous, tissue biopsy, organbiopsy, synovial fluid, urine, bile fluid, cerebrospinal fluid, saliva,mucosal secretion, effusion, sweat or their combination.

The proteins of the sample are in one embodiment distributed on varioussupport matrices by methods specific to each matrix. Suitable matricesmay, in one embodiment be paper, cellulose acetate, silica, glass,carbon, sugars, plastics and derivatives thereof, and a person skilledin the art will be familiar with the techniques of using such supportmatrices for the separation of proteins.

“Marker” in the context of the present invention refers to a polypeptidewhich is differentially present in a sample taken from subjects havinghaving a mTBI, as compared to a comparable sample taken from controlsubjects (e.g., a person with an orthopedic injury or a healthysubject). The term “biomarker” is used interchangeably with the term“marker.”

In one embodiment, determining the expression of a biomarker refers tomethods to assess mRNA abundance, or in another embodiment, gene productabundance. According to this aspect of the invention, and in oneembodiment, gene product refers to the translated protein. In oneembodiment, protein abundance reflects gene expression profiles, whichmay be determined, in other embodiments, by any methods known in theart, such as, but not limited to Western blot analysis, RIA, ELISA,HPLC, functional assays, such as enzymatic assays, as applicable, andothers. In one embodiment, expression profile is determined by a changein mRNA levels, or in another embodiment in surface expression, or inanother embodiment in secretion or in another embodiment otherpartitioning of a polypeptide.

As used herein, the term “independently associated” refers to anassociation that is not necessarily causative, e.g., the level ofexpression of the biomarker or the presence of the biomarker does notcause the disease or adverse condition provided herein.

As used herein, “increased expression” or “increase in level or“elevated level” refer to an increase in the level of a biomarkerrelative to the level or activity of the biomarker in a standard. Anincrease in level may refer to between a 10 to about a 250% increase inbiomarker levels in a biological sample. In another embodiment, theincrease of the biological maker level taken from a mTBI subject is1-10%, 11-20%, 21-30%, 31-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90%,91-150% elevated over the levels of a biomarker taken from a normalsubject (standard).

As used herein, “compared to a standard”, refers to relative changes inbiomarker levels where the standard is derived from a single individual,or is derived from pooled subjects who have been successfullycategorized as being healthy.

As used herein, the term “measuring” refers to methods which includedetecting the presence or absence of marker(s) in the sample,quantifying the amount of marker(s) in the sample, and/or qualifying thetype of biomarker. Measuring can be accomplished by methods known in theart and those further described herein, including but not limited toSELDI and immunoassay. Any suitable methods can be used to detect andmeasure one or more of the markers described herein. These methodsinclude, without limitation, mass spectrometry (e.g., laserdesorption/ionization mass spectrometry), fluorescence (e.g. sandwichimmunoassay), surface plasmon resonance, ellipsometry and atomic forcemicroscopy.

As used herein, the phrase “differentially present” refers todifferences in the quantity and/or the frequency of a marker present ina sample taken from subjects having mTBI.

A polypeptide is differentially present between two samples if theamount of the polypeptide in one sample is statistically significantlydifferent from the amount of the polypeptide in the other sample. Forexample, a polypeptide is differentially present between the two samplesif it is present at least about 120%, at least about 130%, at leastabout 150%, at least about 180%, at least about 200%, at least about300%, at least about 500%, at least about 700%, at least about 900%, orat least about 1000% greater than it is present in the other sample, orif it is detectable in one sample and not detectable in the other.

Alternatively or additionally, a polypeptide is differentially presentbetween two sets of samples if the frequency of detecting thepolypeptide in the subjects' samples is statistically significantlyhigher or lower than in the control samples. For example, a polypeptideis differentially present between the two sets of samples if it isdetected at least about 120%, at least about 130%, at least about 150%,at least about 180%, at least about 200%, at least about 300%, at leastabout 500%, at least about 700%, at least about 900%, or at least about1000% more frequently or less frequently observed in one set of samplesthan the other set of samples.

As used herein, the term “diagnostic” refers to identifying the presenceor nature of a pathologic condition, for e.g., mTBI. As used herein, theterm “sensitivity” of a diagnostic assay refers to the percentage ofdiseased individuals who test positive (percent of “true positives”).Diseased individuals not detected by the assay are “false negatives.”Subjects who are not diseased and who test negative in the assay, aretermed “true negatives.” The “specificity” of a diagnostic assay may becalculated as 1 minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those without the diseasewho test positive. While a particular diagnostic method may not providea definitive diagnosis of a condition, it suffices if the methodprovides a positive indication that aids in diagnosis.

A “test amount” of a marker can refer to an amount of a marker presentin a sample being tested. A test amount can be either in absolute amount(e.g., μg/mL) or a relative amount (e.g., relative intensity ofsignals).

A “diagnostic amount” of a marker can refer to an amount of a marker ina subject's sample that is consistent with a diagnosis of a brain damageseverity or an adverse cardiological condition from an unknown etiologyor as a result of mTBI. A diagnostic amount can be either in absoluteamount (e.g., 1 μg/mL) or a relative amount (e.g., relative intensity ofsignals).

A “control amount” or a “standard” amount of a marker can be any amountor a range of amount, which is to be compared against a test amount of amarker. For example, a control amount of a marker can be the amount of amarker in a healthy subject. A control amount can be either in absoluteamount (e.g., μg/mL) or a relative amount (e.g., relative intensity ofsignals).

In another embodiment, the methods provided herein, comprise proteinlevel (amount) measurements. In another embodiment, the methods providedherein, comprise DNA measurements. In another embodiment, the methodsprovided herein, comprise RNA measurements. In another embodiment, themethods provided herein, comprise mRNA measurements. In anotherembodiment, methods of measuring the expression level of a given proteinused as a biomarker are known to one of average skill in the art. Inanother embodiment, methods of measuring the transcription level of agiven RNA molecule encoding a protein used as a biomarker are known toone of average skill in the art. In another embodiment, methods ofmeasuring the transcription level of a given mRNA molecule encoding aprotein used as a biomarker are known to one of average skill in theart.

Methods for capturing, analyzing, quantifying, etc., biomarkers are knowin the art, can be captured with capture reagents immobilized to a solidsupport, such as any biochip described herein, a multiwell microtiterplate or a resin. Once captured on a substrate, e.g., biochip orantibody, any suitable method can be used to measure a marker or markersin a sample. For example, markers can be detected and/or measured by avariety of detection methods including for example, gas phase ionspectrometry methods, optical methods, electrochemical methods, atomicforce microscopy and radio frequency methods. Using these methods, oneor more markers can be detected. MAP analysis represents a highlyquantitative and rapid method for simultaneously analyzing a largenumber of specific antigens using a very small volume of patient plasma.In another embodiment, analysis of circulating antigen levels within acollected biological sample, via MAP, yields results equivalent to anELISA assay. In another embodiment, MAP yields results with greaterefficiency and with a higher throughput capacity, than an ELISA assay.

If desired, the sample can be prepared to enhance detectability of themarkers. For example, to increase the detectability of markers, a bloodserum sample from the subject can be fractionated by, e.g., Cibacronblue agarose chromatography and single stranded DNA affinitychromatography, anion exchange chromatography, affinity chromatography(e.g., with antibodies) and the like. The method of fractionationdepends on the type of detection method used. Any method that enrichesfor the protein of interest can be used. Sample preparations, such aspre-fractionation protocols, are optional and may not be necessary toenhance detectability of markers depending on the methods of detectionused. For example, sample preparation may be unnecessary if antibodiesthat specifically bind markers are used to detect the presence ofmarkers in a sample.

Typically, sample preparation involves fractionation of the sample andcollection of fractions determined to contain the biomarkers. Methods ofpre-fractionation are well known to those of skill in the art andinclude include, for example, size exclusion chromatography, massspectrometry, ion exchange chromatography, heparin chromatography,affinity chromatography, sequential extraction, gel electrophoresis andliquid chromatography. The analytes also may be modified prior todetection. These methods are useful to simplify the sample for furtheranalysis. For example, it can be useful to remove high abundanceproteins, such as albumin, from blood before analysis. Examples ofmethods of fractionation are described in PCT/US03/00531, but are notlimited to, various kinds of chromatography (e.g., anion exchangechromatography, affinity chromatography, sequential extraction, and highperformance liquid chromatography) and mass spectrometry. The separationand detection of the proteins in a plasma sample generates a proteinspectra for that sample.

Biomarkers in a sample can also be separated by high-resolutionelectrophoresis, e.g., one or two-dimensional gel electrophoresis. Afraction containing a marker can be isolated and further analyzed by gasphase ion spectrometry. In another embodiment, two-dimensional gelelectrophoresis is used to generate two-dimensional array of spots ofbiomarkers, including one or more markers. See, e.g., Jungblut andThiede, Mass Specir. Rev. 16:145-162 (1997).

The two-dimensional gel electrophoresis can be performed using methodsknown in the art. See, e.g., Deutscher ed., Methods In Enzymology vol.182. Typically, biomarkers in a sample are separated by, e.g.,isoelectric focusing, during which biomarkers in a sample are separatedin a pH gradient until they reach a spot where their net charge is zero(i.e., isoelectric point). This first separation step results inone-dimensional array of biomarkers. The biomarkers in one-dimensionalarray are further separated using a technique generally distinct fromthat used in the first separation step. For example, in the seconddimension, biomarkers separated by isoelectric focusing are furtherseparated using a polyacrylamide gel, such as polyacrylamide gelelectrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE).SDS-PAGE gel allows further separation based on molecular mass ofbiomarkers. Typically, two-dimensional gel electrophoresis can separatechemically different biomarkers in the molecular mass range from1000-200,000 Da within complex mixtures. The pI range of these gels isabout 3-10 (wide range gels).

Biomarkers in the two-dimensional array can be detected using anysuitable methods known in the art. For example, biomarkers in a gel canbe labeled or stained (e.g., Coomassie Blue or silver staining). If gelelectrophoresis generates spots that correspond to the molecular weightof one or more markers of the invention, the spot can be furtheranalyzed by gas phase ion spectrometry. For example, spots can beexcised from the gel and analyzed by gas phase ion spectrometry.Alternatively, the gel containing biomarkers can be transferred to aninert membrane by applying an electric field. Then a spot on themembrane that approximately corresponds to the molecular weight of amarker can be analyzed by gas phase ion spectrometry. In gas phase ionspectrometry, the spots can be analyzed using any suitable techniques,such as MALDI or SELDI (e.g., using a PROTEINCHIP® array) as describedherein.

Another method available for use in the present invention is gaschromatography. Prior to gas phase ion spectrometry analysis, it may bedesirable to cleave biomarkers in the spot into smaller fragments usingcleaving reagents, such as proteases (e.g., trypsin). The digestion ofbiomarkers into small fragments provides a mass fingerprint of thebiomarkers in the spot, which can be used to determine the identity ofmarkers if desired.

In one embodiment, the biological sample is analysed for the presence ofthe biomarker(s). In another embodiment, methods for protein analysisthat are well known in the arts and are available for use in the presentinvention include, but are not limited to, Mass Spectrometry,Two-Dimensional Electrophoresis Chromatography High Performance LiquidChromatography, Reversed-Phase Chromatography Ion ExchangeChromatography, and the like.

In another embodiment, an immunoassay can be used to detect and analyzemarkers in a sample. This method comprises: (a) providing an antibodythat specifically binds to a marker; (b) contacting a sample with theantibody; and (c) detecting the presence of a complex of the antibodybound to the marker in the sample.

An immunoassay is an assay that uses an antibody to specifically bind anantigen (e.g., a marker). The immunoassay is characterized by the use ofspecific binding properties of a particular antibody to isolate, target,and/or quantify the antigen. The phrase “specifically (or selectively)binds” to an antibody or “specifically (or selectively) immunoreactivewith,” when referring to a protein or peptide, refers to a bindingreaction that is determinative of the presence of the protein in aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein at least two times the background and do notsubstantially bind in a significant amount to other proteins present inthe sample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to amarker from specific species such as rat, mouse, or human can beselected to obtain only those polyclonal antibodies that arespecifically immunoreactive with that marker and not with otherproteins, except for polymorphic variants and alleles of the marker.This selection may be achieved by subtracting out antibodies thatcross-react with the marker molecules from other species.

In another embodiment, provided herein is a kit for diagnosing orproviding prognosis for a subject developing brain damage as a result ofmTBI, comprising equipment including, but not limited to, assays andanalytical tools for the assays, both as described herein below in theexemplification, reagents, standards and instructions for analyzing theexpression level of two or more biomarkers in a biological sample of thesubject.

Generally, a sample obtained from a subject can be contacted with theantibody that specifically binds the marker. Optionally, the antibodycan be fixed to a solid support to facilitate washing and subsequentisolation of the complex, prior to contacting the antibody with asample. Examples of solid supports include glass or plastic in the formof, e.g., a microtiter plate, a stick, a bead, or a microbead.Antibodies can also be attached to a probe substrate or PROTEINCHIP®array described above. In one embodiment, the sample is a biologicalfluid sample taken from a subject. Examples of biological fluid samplesinclude blood, serum, plasma, nipple aspirate, urine, tears, saliva etc.In another embodiment, the biological fluid comprises blood serum. Thesample can be diluted with a suitable eluant before contacting thesample to the antibody.

After incubating the sample with antibodies, the mixture is washed andthe antibody-marker complex formed can be detected. This can beaccomplished by incubating the washed mixture with a detection reagent.This detection reagent may be, e.g., a second antibody which is labeledwith a detectable label. Exemplary detectable labels include magneticbeads (e.g., DYNABEADS™), fluorescent dyes, radiolabels, enzymes (e.g.,horse radish peroxide, alkaline phosphatase and others commonly used inan ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic beads. Alternatively, the marker in the sample can bedetected using an indirect assay, wherein, for example, a second,labeled antibody is used to detect bound marker-specific antibody,and/or in a competition or inhibition assay wherein, for example, amonoclonal antibody which binds to a distinct epitope of the marker isincubated simultaneously with the mixture.

Methods for measuring the amount of, or presence of, antibody-markercomplex include, for example, detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, birefringenceor refractive index (e.g., surface plasmon resonance, ellipsometry, aresonant mirror method, a grating coupler waveguide method orinterferometry). Optical methods include microscopy (both confocal andnon-confocal), imaging methods and non-imaging methods. Electrochemicalmethods include voltametry and amperometry methods. Radio frequencymethods include multipolar resonance spectroscopy. Methods forperforming these assays are readily known in the art. Useful assaysinclude, for example, an enzyme immune assay (EIA) such as enzyme-linkedimmunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blotassay, or a slot blot assay. These methods are also described in, e.g.,Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai,ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed.1991); and Harlow & Lane, supra.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,marker, volume of solution, concentrations and the like. Usually theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Immunoassays can be used to determine presence or absence of a marker ina sample as well as the quantity of a marker in a sample. The amount ofan antibody-marker complex can be determined by comparing to a standard.A standard can be, e.g., a known compound or another protein known to bepresent in a sample. As noted above, the test amount of marker need notbe measured in absolute units, as long as the unit of measurement can becompared to a control.

When the sample is measured and data is generated, e.g., by methodsdescribed herein such as, but not limited to mass spectrometry, the datais then analyzed by a computer software program. Generally, the softwarecan comprise code that converts signal from the mass spectrometer intocomputer readable form. The software also can include code that appliesan algorithm to the analysis of the signal to determine whether thesignal represents a “peak” in the signal corresponding to a marker ofthis invention, or other useful markers. The software also can includecode that executes an algorithm that compares signal from a test sampleto a typical signal characteristic of “normal” one and determines thecloseness of fit between the two signals. The software also can includecode indicating which the test sample is closest to, thereby providing aprobable diagnosis.

In one embodiment of the present invention, multiple biomarkers aremeasured. The use of multiple biomarkers increases the predictive valueof the test and provides greater utility in diagnosis, categorization ofthe severity of a subject or patient's condition, patient stratificationand patient monitoring. The process called “Pattern recognition” detectsthe patterns formed by multiple biomarkers and greatly improves thesensitivity and specificity of clinical proteomics for predictivemedicine. Subtle variations in data from clinical samples, e.g.,obtained using methods provided herein and those know in the art,indicate that certain patterns of protein expression can predictphenotypes such as the presence or absence of a certain disease, aparticular stage of a disease, or a positive or adverse response to drugtreatments.

As used herein, a protein may have “homology” or be “homologous” toanother protein if the two proteins have similar amino acid sequencesand have similar biological activities or functions. A protein from twodifferent organisms may have homology or be homologous when the encodedamino acid sequences of the proteins are similar and the proteins have asimilar biological activity or function. It is understood that“homologous” does not necessarily imply that there is an evolutionaryrelationship between the proteins. In one embodiment, a homologousprotein exhibits 50% sequence similarity to the wild type protein, or inanother embodiment 60% sequence similarity, or in another embodiment 70%sequence similarity. or in another embodiment 80%, 85% or 90% sequencesimilarity to the wild type protein. or in another embodiment, ahomologous protein exhibits 95%, 97%, 98% or 99% sequence similarity.

In one embodiment, the methods of the invention provide for the use ofmultiple assays, to evaluate differential gene expression. In anotherembodiment, arrays are used since microarray analysis allows in anotherembodiment simulataneous gene expression analysis of multiple genes in ahigh-throughput mode.

In one embodiment, a combination of biomarkers may provide greaterpredictive value than single markers alone. In another embodiment, thedetection of a plurality of markers in a sample increases the percentageof true positive and true negative diagnoses and would decrease thepercentage of false positive or false negative diagnoses. Thus, themethods of the present invention can include the measurement of morethan one biomarker.

In other embodiments, the measurement of markers can involve quantifyingthe markers to correlate the detection of markers with a probablediagnosis of the mTBI or brain damage, as described herein.

The correlation may take into account the amount of the marker ormarkers in the sample compared to a control amount of the marker ormarkers (up or down regulation of the marker or markers) (e.g., innormal healthy subjects). A control can be, e.g., the average or medianamount of marker present in comparable samples of healthy subjects. Thecontrol amount is measured under the same or substantially similarexperimental conditions as in measuring the test amount. The correlationmay take into account the presence or absence of the markers in a testsample and the frequency of detection of the same markers in a control.The correlation may take into account both of such factors to facilitatedetermining/practicing the methods provided herein.

In another embodiment, a suitable statistical tool, known to one ofskill in the art, is used to determine the level of a biomarker relativeto a standard.

In one embodiment, continuous measures are described using simplestatistics (mean, median, standard deviation, and range) andcategorical/ordinal data (e.g. race, gender, and remodeling geometry)with tables and frequencies.

In another embodiment, graphical methods including histograms, scatterplots, and box plots are used to understand aspects of data quality andexamine assumptions that underlie parametric and semi-parametric models.

In one embodiment, to better understand the changes in exposure andoutcome over time, individual trajectories are plotted as well as groupsummaries across time, and Kaplan-Meier plots are used to estimatesurvival probabilities.

In one embodiment the methods provided herein further comprise managingsubject treatment based on the status. Such management describes theactions of the physician or clinician subsequent to determining theseverity of brain damage. For example, if the result of the methods ofthe present invention is inconclusive or there is reason thatconfirmation of status is necessary, the physician may order more tests.Alternatively, if the status indicates that treatment is appropriate,the physician may schedule the patient for treatment. Likewise, if theresult is negative, e.g., the status indicates no need for braindamagetreatment is needed, no further action may be warranted.Furthermore, if the results show that treatment has been successful, nofurther management may be necessary. The invention also provides forsuch methods where the biomarkers (or specific combination ofbiomarkers) are measured again after subject management. In these cases,the methods are used to monitor the status of the severity of braindamage in a subject. Because of the ease of use of the methods and thelack of invasiveness of the methods, the methods can be repeated aftereach treatment the patient receives. This allows the physician to followthe effectiveness of the course of treatment. If the results show thatthe treatment is not effective, the course of treatment can be alteredaccordingly. This enables the physician to be flexible in the treatmentoptions.

In another example, the methods for detecting markers can be used toassay for and to identify compounds that modulate expression of thesemarkers in vivo or in vitro.

In yet another embodiment, the markers are used in heredity studies todetermine if the subject is at risk for developing a more severe case ofbrain damage.

“Solid support” refers to a solid material which can be derivatizedwith, or otherwise attached to, a capture reagent. Exemplary solidsupports include probes, microtiter plates and chromatographic resins.

“Probe” in the context of this invention refers to a device adapted toengage a probe interface of a gas phase ion spectrometer (e.g., a massspectrometer) and to present an analyte to ionizing energy forionization and introduction into a gas phase ion spectrometer, such as amass spectrometer. A “probe” will generally comprise a solid substrate(either flexible or rigid) comprising a sample presenting surface onwhich an analyte is presented to the source of ionizing energy.

“Eluant” or “wash solution” refers to an agent, typically a solution,which is used to affect or modify adsorption of an analyte to anadsorbent surface and/or remove unbound materials from the surface. Theelution characteristics of an eluant can depend, for example, on pH,ionic strength, hydrophobicity, degree of chaotropism, detergentstrength and temperature.

“Analyte” refers to any component of a sample that is desired to bedetected. The term can refer to a single component or a plurality ofcomponents in the sample.

The “complexity” of a sample adsorbed to an adsorption surface of anaffinity capture probe means the number of different protein speciesthat are adsorbed. “Monitoring” refers to recording changes in acontinuously varying parameter.

In one embodiment, provided herein is a kit comprising reagents fordetecting the biomarker levels, wherein the reagents may includeantibodies, nucleic acids, which may hybridize to mRNA isolated from abiological sample, and the like. In one embodiment, reagents may belabelled, or in another embodiment nucleic acids isolated from abiological sample are labelled. In another embodiment, the kit providesinstructions for detecting the label qualitatively in anotherembodiment, quantitatively.

In another embodiment the kit further comprises a buffering agent, or inanother embodiment, a preservative, or in another embodiment a proteinstabilizing agent. In one embodiment, the kit further comprises anenzyme or a substrate. In one embodiment, the substrate may be a meansof detecting a label, or in another embodiment the expressed proteinproduct itself. In one embodiment, the kit further comprises reagentsthat are necessary for detection of nucleic acids, amino acids orhybridization signals for nucleic acids.

In one embodiment, detecting differential expression of the genes viathe kits of the invention is accomplished using established PCR, ELISA,RIA, and other similarly recognized methods, and the reagents comprisethose appropriate for the particular assay for detection.

In one embodiment, the results obtained are compared to a standard,which, in another embodiment, may comprise a series of standards, which,in another embodiment is used in the kits of the invention forquantification of differential levels of the biomarker or differentialexpression. In one embodiment, the standard may comprise any embodimentlisted herein, and in another embodiment, will be suitable for aparticular application of the kit. In one embodiment, the standardcomprises antibodies for detecting a standard biomarker. In oneembodiment, the standard comprises nucleic acids when the kit is usedfor the determination of nucleic acid profile, or in another embodimentthe standard is a protein when the kit is used for the determination ofexpressed protein profile.

In one embodiment, the kit may be adapted for high-throughput screening,and comprise a microarray.

In one embodiment, the kit further comprise agents, which in anotherembodiment may comprise antibodies, or other agents which detectactivity or in another embodiment expression of the translated proteinproduct. In one embodiment the agents comprise antibodies that detectthe presence of specific nucleic acids.

In one embodiment, the kit comprises a microarray, which comprises cRNAof the genes indicated, and others. In one embodiment, the kit maycomprise standard oligonucleotide probes, PCR reagents and detectablelabels. In another embodiment, the kit may comprise biological samplestaken from human subjects. The standard will comprise all embodimentslisted herein for the standard, including in one embodiment nucleic acidfrom pooled samples as provided herein.

In one embodiment, the kit further comprises a positive and negativecontrol, wherein said standards can be assayed and compared to the testsample.

In one embodiment, the kit may further comprise labeled cDNA.Fluorescently labeled cDNA probes may be generated through incorporationof fluorescent nucleotides by reverse transcription of RNA extractedfrom samples of interest Such methods have been shown to have thesensitivity required to detect rare transcripts, which are expressed ata few copies per cell, and to reproducibly detect at least approximatelytwo-fold differences in the expression.

In one embodiment, the methods of this invention employ probes andprimers, which may include repetitive stretches of adenine nucleotides(poly-A tails) normally attached at the ends of the RNA, for theidentification of differentially expressed genes. In another embodiment,kits of this invention may comprise such probes.

In one embodiment, the biomarker is a functional biomarker or afunctional fragment thereof. In another embodiment, the biomarker is afunctional variant or fragment thereof of a biomarker provided herein.In another embodiment the biomarker is a homolog of a biomarker providedherein, where in another embodiment, it is a paralog or an ortholog of abiomarker provided herein.

In one embodiment, cRNA refers to complementary ribonucleic acid orsubstantially complementary ribonucleic acid. In another embodiment,cRNA refers to the hybridization or base pairing between nucleotides ornucleic acids, such as, for instance, between the two strands RNAmolecule or between an oligonucleotide primer and a primer binding siteon a single stranded nucleic acid to be sequenced or amplified.Complementary nucleotides are, generally, A and T (or A and U), or C andG. Two single stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andcompared and with appropriate nucleotide insertions or deletions, pairin one embodiment, with at least about 70% of the nucleotides of theother strand, or in another embodiment with about 90% to 95%, and inanother embodiment with about 98 to 100%. The invention also provides amethod for treating a mild traumatic brain injury (mTBI) in a subjecthaving a negative computed tomography (CT) test result. The methodincludes the prognosis or diagnosis of said mTBI of the invention and,based on the prognosis or diagnosis, treating said mTBI in said subject.

As used herein, the term “treating” may encompass curing, preventing,reducing the incidence of, ameliorating symptoms of, to inducingremission of, or slowing the progression of a disease. The terms“reducing”, “suppressing” and “inhibiting” refer to lessening ordecreasing.

The term “about” as used herein means in quantitative terms plus orminus 5%, or in another embodiment plus or minus 10%, or in anotherembodiment plus or minus 15%, or in another embodiment plus or minus20%.

The term “subject” refers to a mammal, including a human, in need oftherapy for, or susceptible to, a condition or its sequelae. The term“subject” does not exclude an individual that is normal in all respects.The term “patient” is encompassed by the term “subject”.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLES

Materials and Methods

Study Participants.

The Institutional Review Boards of the University of Pennsylvania andTexas Medical Center, Houston, reviewed and approved the study. Allparticipants in this study provided written informed consent (or assentif written consent was given by the minor's parent) and were recruitedand assessed with approval from and according to the ethical guidelinesof the Institutional Review Boards of the recruiting institutions. Allprocedures were conducted in accord with the ethical standards of theHelsinki Declaration of 1975, as revised in 2000.

This neurodegeneration biomarker study examined 38 participants withplasma collected within 24 hours of injury. Of those, 17 sustained amTBI, 13 sustained an orthopedic injury (OI) and 8 were uninjuredcontrols (UC). This effort was part of a larger study (n=205) comprisingright-handed participants of ages 12-30 years, who were recruited andtested on neuropsychological and brain imaging measures at baseline(within 96 hours of injury), and at follow-up sessions at 1 month(neuropsychological measures only) and 3 months. Participant recruitmentwas from an unselected series of patients admitted to emergency centersin the Texas Medical Center, Houston, including Ben Taub GeneralHospital, Texas Children's Hospital, and Memorial Herman Hospital, or,for the uninjured control group, from the greater Houston metropolitanarea. The smaller biomarker study group did not differ significantlyfrom the larger study sample on age, socioeconomic status (SES), race,gender, or extracranial Injury Severity Score (ISS).

The 17 participants providing plasma samples with mTBI, as defined bycriteria from the Centers for Disease Control, had an injury to the headfrom blunt trauma, acceleration, or deceleration forces with one or moreof the following conditions: (1) observed or self-reported contusion,disorientation, or impaired consciousness, dysfunction of memory at thetime of the injury, loss of consciousness lasting less than 30 minutes;and (2) symptoms such as headache, dizziness, fatigue, irritability, andpoor concentration soon after the injury. Additional inclusion criteriawere a Glasgow Coma Scale score of 13-15 upon examination at anemergency center, no abnormal findings on head CT, duration of loss ofconsciousness for no more than 30 minutes, post-traumatic amnesia forless than 24 hours, and an Abbreviated Injury Score (AIS)≤3 and an ISSof ≤12 modified to exclude the head region. Comparator participants wereof two cohorts. For one, participants with 01 were recruited less than96 hours post-injury provided they met the following criteria:right-handed, 12-30 years old, no loss of consciousness, nopost-traumatic amnesia, no overt intracranial injury, AIS<3 for anyregion of the body and an ISS 12, and a normal brain CT (if done). Asecond UC cohort consisted of 8 healthy participants who had notsustained any injury, but were similar to the two injury groups in age,gender and level of education.

Exclusions included non-fluency in either English or -Spanish, failureto provide adequate contact information for scheduling follow-upassessments, blood alcohol level>200 mg/dL, previous hospitalization forhead injury, pregnancy when screened prior to brain imaging,pre-existing neurologic disorder associated with cerebral dysfunctionand/or cognitive deficit (e.g., cerebral palsy, mental retardation,epilepsy) or diagnosed dyslexia, pre-existing severe psychiatricdisorder (e.g., bipolar disorder, schizophrenia), and contraindicationsto undergoing MRI. The OI comparison group was included to control forrisk factors that predispose to injury, including preexisting behavioralproblems, learning disabilities, and family variables, along with ageneral trauma context similar to those with mTBI. The uninjured groupwas included to examine effects not due to injury and to compare injuredpatients to the general young adult population. All participantsprovided written informed consent (or assent if written consent wasgiven by the minor's parent) and were recruited and assessed withapproval from and according to the ethical guidelines of the recruitinginstitutions.

Neurobehavioral Assessments.

Participants were administered tests of cognition and assessed forsymptoms related to post-concussive injury. For comparison withneurodegeneration biomarker findings, we analyzed data from threedomains, speed of processing, executive memory and cognitiveflexibility, along with symptoms of concussion. The analyses wereconducted by investigators blinded to the plasma biomarker data.

Rivermead Post Concussion Symptoms Questionnaire (RPCS).

The RPCS is a 16-item self-report of cognitive, emotional, and somaticcomplaints that are commonly reported following mTBI. Factor analyseshave elicited a 3-factor solution comprising cognitive, somatic, andemotional problems, although different factor structures have beenreported. The participants were asked to rate the severity of eachsymptom (currently compared to pre-injury levels) from 0—‘notexperienced at all’ to 4—‘severe problem.’ The primary variable was thetotal score.

Symbol-Digit Modalities Test (SDMT).

This is a timed substitution task with written and oral responsemodalities and is highly sensitive to processing speed deficits in the8-78 year age range. Using a reference key, each examinee was asked topair specific numbers with given geometric symbols within 90 seconds.The number of correct responses in the written modality was the variableused in this study.

KeepTrack Task (KT).

This updating task requires adding and deleting items in working memoryaccording to semantic category, and the maintenance of semanticcategorical representations. It has been validated in the mild TBIpopulation. The variable used was the mean percent correct items perlist recalled.

Diffusion Tensor Imaging.

All participants underwent MRI without sedation on a Philips 3.0 TeslaAchieva scanner. Rigorous quality assurance testing was performedincluding American College of Radiology phantom testing: no concernswith quality assurance were noted during the course of the study.

An axial single-shot spin-echo echo-planar imaging sequence with 30diffusion-encoding directions was used for DTI acquisition. Otherparameters included a 256 mm field of view, an acquisition voxel size of2×2×2 mm³, repetition time of 11526 ms, echo time of 51 ms, sensitivityencoding (SENSE) reduction factor of 2, two B factors (0 s/mm² low B,and 1000 s/mm high B), with two acquisitions to average the signal ofthe two DTI scans in order to ensure better signal-to-noise ratio. DTIacquisition consisted of 70 slices. A SENSE 8-channel head coil wasused.

Image Processing

The corpus callosum, right and left uncinate fasciculi, and right andleft frontal lobes were selected as structures of interest due to theirknown vulnerability in DTI studies of TBI and their presumed relation tothe measures of speed of cognitive processing, memory updating, andexecutive function, and post-concussion symptoms. Additionally, DTImeasurement of these structures has been shown to be reproducible bothbetween and within raters on quantitative tractography using previouslypublished protocols. In this study, DTI data were analyzed twice by asingle rater to establish intra-rater reliability using intra-classcorrelational coefficients (ICCs). A subset of the images was analyzedby two raters to establish inter-rater reliability. ICCs for allmeasurements were above 0.95.

Quantifying the Neurode Generation Biomarker SNTF

The sandwich immunoassay for quantifying calpain-cleaved αII-spectrinN-terminal fragment (SNTF) from human plasma is a modification of amethod published previously, in which the enzymatic amplification anddetection steps of ELISA were replaced with electrochemiluminescencedetection chemistry. Briefly, 96 well plastic microplates with anunderside electrode (Meso Scale Discovery) were coated overnight withthe capture antibody, a monoclonal directed at the SH3 domain in theN-terminal portion of the a-spectrin subimit (D8/B7 @ 1/1,000; Abcam).For the antigen capture step, human plasma samples diluted to 40% orSNTF standards (25 μLs/well) prepared in 0.25% bovine serum albumin inTris-buffered saline (pH 7.4) were added in triplicate for 2 hours at22° C. The detecting antibody was a purified rabbit IgG prepared in ourlaboratory reactive with the calpain-generated neoepitope at thecarboxyl-end of the calpain-derived a-spectrin ˜150 kDa amino-terminalfragment (SNTF; 1/5,000). The specificity of this cleavage site-specificantibody for SNTF has been well established. The reporter probe was goatanti-rabbit IgG conjugated to ruthenium (Sulfotag, Meso Scale Discovery,Rockville, Md.; 1/500). In the presence of read buffer containingtripropylamine and application of current to the plate electrode, achemiluminescent product is produced in proportion to the bound antigen.Chemiluminescent signals were quantified by a SECTOR Imager 2400 system(Meso Scale Discovery). Standard curves were generated using serialdilution of a preparation of α-spectrin partially purified from brainand digested with purified calpain I. Briefly, the digestion wasperformed for 10 minutes at 30° C. at a 300:1 ratio by weight ofspectrin extract:calpain I in a buffer of 5 mM Tris-HCl (pH 7.8), 0.6 MKCl, 5 mM β-mercaptoethanol, 2 mM CaCl₂. Purified bovine erythrocytecalpain I for the digest was obtained from Sigma (St. Louis, Mo.).Reactions were quenched and the calpain I inactivated by addition of 5mM EDTA followed by freeze-thaw.

One unit of SNTF is defined as the signal derived from the SNTF standarddiluted to 1 nanoliter per ml, corresponding to ˜500 pg of thespectrin-containing brain extract starting material per ml. The minimumreliable detection sensitivity was 10 units.

Control experiments were performed to distinguish SNTF-related signalsfrom non-specific signals emanating from heterophilic substances thatare present in a subset of human plasma samples and confound attempts tomeasure very small amounts of target antigen.

These control immunoassays were conducted as above, except that thedetecting IgG specific for SNTF was replaced with normal IgG purifiedfrom pre-immune serum from the same rabbit. SNTF-specific signals werecalculated as the difference between the specific and pre-immunedetecting IgG signals and converted to standardized units. Theimmunoassays were conducted and analyzed by investigators blinded to allother patient data.

Results

Example 1 Changes in Long-Term Cognitive Function in a Subset of mTBICases

A total of 38 participants provided plasma samples on the day of injuryfor quantification of the neurodegeneration biomarker SNTF: 17 werediagnosed with mTBI and 13 with orthopedic injury (OI), whereas 8 wereuninjured controls (UC). The biomarker study subgroup did not differfrom the overall study group in terms of initial injury severity, age,gender, or other factors (Table 1).

Among these cases, brain structural integrity was assessed by DTI within4 days of injury for 28 of the participants, and brain performance wasevaluated by neuropsychological testing within 4 days of injury and at 1and 3 months thereafter for 27-29 of the participants, depending on thetest battery. The three cohorts did not differ significantly from oneanother in age, gender, or level of education.

In comparison with OI and UC groups, the mTBI group demonstrated overallperformance deficits at 3 months post-injury on the Symbol DigitModalities Test (SDMT), KeepTrack (KT), and Rivermead Post-ConcussionSymptoms Questionnaire (RPCS) cognitive, emotional, and somaticsubscales, similar to reports from prior studies. Neuropsychologicaltest performance varied widely among the mTBI participants: someperformed indistinguishably from the UC group at both early and latetime points, while other participants showed impairments at the acuteand/or 1 month time point that resolved by 3 months, and a third setexhibited dysfunction persisting out to 3 months.

Example 2 Plasma SNTF is Elevated in a Subset of mTBI Cases

We evaluated SNTF as a candidate plasma biomarker for human mTBI. Thisα-spectrin fragment is generated by the calpain family of cysteineproteases and accumulates in axons damaged by stretch injury in vitro orTBI in vivo. It is released from neurons upon plasma membranedisruption. SNTF has not been evaluated before as a prognostic marker inmTBI. Here, plasma SNTF measured on the day of injury was above thelower limit of detection of 10 units in an ultrasensitive sandwichimmunoassay in a subset of participants: 7 of 17 mTBI cases and 3 of 13OI cases. In contrast, plasma SNTF was below the lower limit ofdetection in all 8 UC participants. The immunoassay signals from thepositive plasma samples were confirmed as being specific for SNTF, andnot from heterophilic substances that can confound human plasmabiomarker studies, by control experiments in which the SNTF-specificdetecting IgG was replaced with pre-immune IgG isolated from the samerabbit. The SNTF-positive mTBI participants were both male and femaleand their injuries spanned a variety of mechanisms from sports, assault,motor vehicle/motorcycle crashes, falls, and being struck by a fallingobject. Among the SNTF positive participants, the plasma sampling timeranged broadly from 1-24 hours post-injury, and the absolute SNTF levelsranged from 20-150 units. The SNTF positive and negative groups did notdiffer significantly from one another in age or gender.

Example 3 Elevated Plasma SNTF on the Day of Injury is Related to WhiteMatter Damage and Long-Term Cognitive Dysfunction

To examine the relationship between plasma SNTF levels on the day ofmTBI and DAI, the 28 participants among the mTBI, 01, and UC cohortswith usable neuroradiological data were dichotomized as either SNTFpositive or negative, and the two groups were evaluated comparativelyfor axon tract structural abnormalities by DTI. Compared with the 19SNTF negative cases analyzed by DTI within 4 days of injury, the 9 SNTFpositive cases exhibited significant reductions in FA and increases inADC in the corpus callosum and uncinate fasciculus (p<05; Table 2). TheFA and ADC are thought to quantify the orientation and structuralintegrity of white matter, and their differences as a function ofdichotomized plasma SNTF levels provide evidence that plasma elevationsin this neurodegeneration biomarker after injury may be related to DAI.

TABLE 1 Representativeness of biomarker study subgroup relative toparticipants in the ongoing mTBI study. Biomarker Group Overall GroupMean Mean P- (+/−S.D.) (n = 205) (+/−S.D.) (n = 38) value Age atBaseline 20.2 (+/−5.4) 20.5 (+/−5.8) 0.80 SES −0.0028 (+/−0.79) −0.039(+/−0.72) 0.80 Race % non Black 61 60 0.87 Gender % Female 33 26 0.38GCS (mTBI) % <15 23 24 0.85 Noncranial Injury 0.93 (+/−1.17) 1.37(+/−1.42) 0.13 Severity

There were no differences related to demographics or injury between thebiomarker study group and the overall study group (t-test).

TABLE 2 Plasma SNTF is related to diffusion tensor imaging differencesin select white matter tracts. Mean (SD) Mean (SD) All SNTF− All SNTF+ PEffect Region/metric (n = 19) (n = 9) value Size Corpus callosum FA0.496 (0.02) 0.479 (0.01) 0.034 0.91 ADC 0.821 (0.03) 0.839 (0.02) 0.130.63 Uncinate Fasciculus, Left FA 0.405 (0.02) 0.388 (0.02) 0.09 0.73ADC 0.754 (0.03) 0.775 (0.03) 0.14 0.63 Uncinate Fasciculus, Right FA0.389 (0.01) 0.367 (0.02) 0.001 1.48 ADC 0.774 (0.02) 0.798 (0.03) 0.0350.89 Frontal Lobes, Left FA 0.394 (0.02) 0.383 (0.02) 0.26 0.47 ADC0.765 (0.02) 0.782 (0.02) 0.07 0.77 Frontal Lobes, Right FA 0.382 (0.03)0.381 (0.02) 0.95 0.03 ADC 0.783 (0.02) 0.794 (0.02) 0.15 0.59

Dichotomized plasma SNTF levels on the day of injury discriminate groupson brain white matter structural integrity indexed by diffusion tensorimaging performed within 96 hours. Effect size is reported as Cohen's d,where 0.2-0.49 reflects small, 0.5-0.79 medium, and 0.8 or higher largeeffect size, and P value is from two-tailed t-test.

Long-term behavioral studies have provided evidence that a subset ofCT-negative patients with mTBI develop brain functional disability thatcan persist for many months post-injury. To examine the prognosticrelationship between plasma SNTF levels measured on the day of mTBI andlong-term brain function, participants were evaluated within 4 days andagain at 1 and 3 months post-injury on a battery of tests for cognitiveperformance and assessed for post-concussion symptoms. These includedthe Symbol-Digit Modalities Test (SDMT), which measures speed ofcognitive processing and is a sensitive index of cognitive functioningindependent of intelligence level, the KeepTrack Task, a measure ofmemory updating and executive function, and the RivermeadPost-Concussiom Symptoms Questionnaire (RPCS), a self-report assessmentof the severity of somatic, emotional and cognitive symptoms afterconcussion. For groups dichotomized with respect to plasma SNTF levelson the day of injury, there were marked differences in functionalmeasures at both the acute and long-term time points. Plasma SNTF didnot discriminate symptomatology on the overall RPCS, but showed anassociation with impairments in the cognitive and somatic components at3 months post-injury that did not reach statistical significance. Mostimportantly, a detectable level of plasma SNTF on the day of injurydiscriminated test performance at 3 months on the written versions ofthe SDMT and the KeepTrack task, and the relationship with the formercognitive deficit was highly significant (p<0.01; Table 3).

The significant discrimination in the written and oral versions of theSDMT observed across all study participants based on dichotomized plasmaSNTF (Table 3) was even stronger among the mTBI cases by themselves(written SDMT: SNTF+=46.8; SNTF−=59.1; p=0.011; oral SDMT: SNTF+=70.1;SNTF−=53.3; p=0.024).

Plasma SNTF on the day of mTBI also correlated with recovery ofcognitive performance. Among the 13 mTBI participants evaluated by theoral SDMT in both the acute (1-4 days) and long-term (3 month)post-injury time periods, test scores for the SNTF—cases improved by 17points (+/−5.7, s.e.m.), whereas those for the SNTF+ cases worsened by2.6 points (+/−2.7). The difference in 3 month recovery of cognitiveperformance as a function of dichotomized plasma SNTF levels wassignificant (p<0.03). Six of eight SNTF− cases of mTBI showedimprovement in cognitive performance over 3 months of 5 points orgreater on the oral SDMT, compared with none of the five SNTF+ cases(FIG. 1 ). Based on this preliminary post-hoc assessment, plasma SNTF onthe day of mTBI showed 100% sensitivity and 75% specificity forpredicting failure to improve cognitive performance over the first 3months after a CT-negative mTBI.

TABLE 3 Plasma SNTF on the day of a mTBI relates to impaired cognitiveperformance at 3 months post-injury. Test All SNTF+ All SNTF− Effectsize Symbol-Digit Modalities 52.00 (12.1)  63.47 (14.86) 0.88 Test,Written (total (large) correct responses) KeepTrack Task 88.89 (7.8)  92.72 (5.6)  0.63 (Percent correct recalled) (mod-large) RiverMead 9.44(10.89)  6.37 (11.08) 0.28 Post-Concussion (small) Symptoms (totalscore)

Dichotomized plasma SNTF levels on the day of injury (+/−SD inparentheses) are related to behavioral differences 3 months post-injury.Effect size is reported in Cohen's d, where 0.2-0.49 reflects a small,0.5-0.79 a medium, and 0.8 or higher a large effect size. The differencein cognitive performance on the written Symbol Digit Modalities Testacross all study participants based on plasma SNTF is significant bytwo-tailed t-test (p<0.04), as is the difference within the mTBI casesby themselves (SNTF+=46.8; SNTF−=59.1; p<0.025).

TABLE 4 Dichotomized plasma SNTF levels on the day of injury correlatewith impaired cognitive performance at 3 months. Mean Mean (SEM) (SEM)Cognitive performance SNTF SNTF P test negative positive Value SDMT at 3months All study subjects 63.4 (3.4) 52.0 (4.0) 0.039 mTBI cases 59.1(2.8) 46.8 (2.9) 0.011 ΔSDMT over 3 months mTBI cases +17.5 (5.7)  −2.6(2.7) 0.029

The SDMT scores were significantly worse for the biomarker positivecases both across all study participants and among the mTBI cases bythemselves (two-tailed t-test). In the mTBI group, elevated SNTF on theday of injury also predicted failure to improve cognitive performanceover 3 months.

In this study, we provide evidence that the blood level of theneurodegeneration biomarker SNTF identifies mTBI patients on the day oftheir injury likely to have both white matter changes with advancedneuroimaging suggestive of DAI, and also cognitive dysfunction thatpersists for at least 3 months.

In contrast to the prior findings with other marker candidates, ourresults indicate that the blood level of SNTF and potentially otherneurodegeneration biomarkers sampled in the acute period afterCT-negative mTBI help identify at an early and treatable stage a subsetof cases at risk of developing white matter tract structural damage andlong-term disability.

The injury-induced elevation in plasma SNTF in a subset of mTBI casesreported here show that functionally impactful mTBI triggers calpainactivation and spectrin degradation within vulnerable axons, followed byefflux of the stable fragment SNTF into the brain parenchyma andbloodstream in association with the axon tract damage underlying brainfunctional impairment.

Increased plasma SNTF post-concussion is related not only to structuralevidence for diffuse axonal injury (DAI), but also functional evidencefor long-term cognitive impairment. Whereas a subset of the participantswith mTBI exhibit no discernible deficits on a battery of cognitive,somatic, or emotional tests post-injury, a second group showsperformance deficits that resolve over time, while a third groupdevelops impaired brain performance persisting for at least 3 monthspost-injury. Strikingly, the dichotomized plasma level of SNTF measuredon the day of injury is related to cognitive dysfunction at 3 months, asevidenced by a significant deficit in the SNTF-positive group in theSymbol Digit Modalities Test and trends toward impairments in theKeepTrack test (Table 3) and the cognitive component of the RivermeadPost Concussion Symptoms Questionnaire (RPCS). The ability of plasmaSNTF elevations to significantly differentiate long-term cognitivedecline holds across all 28 participants in the mTBI, OI, and UC groupsand even more strongly among the mTBI cases by themselves. Plasma SNTFon the day of mTBI also discriminated subsequent change in cognitiveperformance on the Symbol Digit Modalities Test, with a positive SNTFfinding predicting failure to improve cognitive performance over 3months post-injury (FIG. 1 ).

Overall, the results show that the blood level of SNTF on the day of aCT-negative mTBI can identify a subset of patients at risk of whitematter damage and persistent disability. SNTF can have prognostic anddiagnostic utilities in the assessment and treatment of mTBI.

Having described the embodiments of the invention with reference to theaccompanying drawings, it is to be understood that the invention is notlimited to the precise embodiments, and that various changes andmodifications may be effected therein by those skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

What is claimed is:
 1. A method for diagnosing and treating a computedtomography (CT)-negative mild traumatic brain injury (mTBI) in asubject, said method comprising: (a) obtaining a blood, serum, or plasmasample from said subject within 24 hours of the injury; (b) contactingsaid sample with an antibody that specifically binds to acalpain-generated neoepitope of calpain-cleaved all-spectrin N-terminalfragment (SNTF) to form an antibody-SNTF complex; (c) measuring theamount of the antibody-SNTF complex, to determine SNTF concentration inthe subject's serum or plasma; (d) comparing said serum or plasmaconcentration of SNTF in the subject to that of a pre-determinedstandard; (e) diagnosing said mTBI in said subject, wherein an elevatedSNTF level relative to the standard indicates the severity of mTBI; and(f) treating said mTBI in said subject.
 2. The method of claim 1,wherein said standard is determined by measuring SNTF concentration in asubject having sustained an orthopedic injury or in a normal uninjuredsubject.
 3. The method of claim 1, wherein said standard is determinedby measuring SNTF concentrations in a subject or pool of subjects havingsustained an orthopedic injury or in a normal uninjured subject or poolof subjects.
 4. The method of claim 1, further comprising the step ofassessing the subject by diffusion tensor imaging (DTI).
 5. The methodof claim 4, wherein said diffusion tensor imaging (DTI) measures thefractional anisotropy (FA) and the apparent diffusion coefficient (ADC).6. The method of claim 1, further comprising the step of assessing thesubject by diffusion tensor imaging (DTI) and by neurobehavioralanalyses.
 7. The method of claim 1, wherein said mTBI is a concussion.8. The method of claim 1, wherein said mTBI is caused by a head injury.9. The method of claim 8, wherein said head injury is blunt trauma,acceleration, or deceleration forces.
 10. The method of claim 1, furthercomprising the steps of: (g) obtaining a blood, serum, or plasma samplefrom said subject, during or after treatment of said subject for mTBI;(h) repeating steps (b)-(d), for the sample obtained during or aftertreatment of said subject; and (i) monitoring the response to thetreatment in the subject having suffered from mTBI, wherein decreasingSNTF levels indicates a subsequent decreased risk of brain damage,long-term functional disability, or long-term neurological dysfunction.11. A method for identifying and treating a subject at risk of sufferingfrom a mild traumatic brain injury (mTBI)-associated abnormality inwhite matter structure or a long-term dysfunction, said methodcomprising: (a) obtaining a blood, serum, or plasma sample from saidsubject within 24 hours of the injury; (b) contacting said sample withan antibody that specifically binds to a calpain-generated neoepitope ofcalpain-cleaved all-spectrin N-terminal fragment (SNTF) to form anantibody-SNTF complex; (c) measuring the amount of the antibody-SNTFcomplex, to determine SNTF concentration in the subject's serum orplasma; (d) comparing said serum or plasma concentration of SNTF in thesubject to that of a pre-determined standard; (e) identifying a subjectat risk of suffering from a mild traumatic brain injury(mTBI)-associated abnormality in white matter structure or a long-termdysfunction, wherein an elevated SNTF level relative to the standardindicates an elevated risk of suffering from a mild traumatic braininjury (mTBI)-associated abnormality in white matter structure or along-term dysfunction; and (f) treating said mTBI in said subject. 12.The method of claim 11, wherein said standard is determined by measuringSNTF concentration in a subject having sustained an orthopedic injury orin a normal uninjured subject.
 13. The method of claim 11, furthercomprising the steps of: (g) obtaining a blood, serum, or plasma samplefrom said subject, during or after treatment of said subject for mTBI;(h) repeating steps (b)-(d), for the sample obtained during or aftertreatment of said subject; and (i) monitoring the response to thetreatment in the subject having suffered from mTBI, wherein decreasingSNTF levels indicates a subsequent decreased risk of brain damage,long-term functional disability, or long-term neurological dysfunction.14. A method for providing a prognosis for and treating a subject havingsuffered from a computed tomography (CT)-negative mild traumatic braininjury (mTBI), said method comprising: (a) obtaining a blood, serum, orplasma sample from said subject within 24 hours of the injury; (b)contacting said sample with an antibody that specifically binds to acalpain-generated neoepitope of calpain-cleaved all-spectrin N-terminalfragment (SNTF) to form an antibody-SNTF complex; (c) measuring theamount of the antibody-SNTF complex, to determine SNTF concentration inthe subject's serum or plasma; (d) comparing said serum or plasmaconcentration of SNTF in the subject to that of a pre-determinedstandard; (e) providing a prognosis for said subject having sufferedfrom said mTBI, wherein an elevated SNTF level relative to the standardindicates an elevated risk of long-term neurological dysfunction of themTBI evolving to brain damage; and (f) treating said mTBI in saidsubject.
 15. The method of claim 14, further comprising the steps of:(g) obtaining a blood, serum, or plasma sample from said subject, duringor after treatment of said subject for mTBI; (h) repeating steps(b)-(d), for the sample obtained during or after treatment of saidsubject; and (i) monitoring the response to the treatment in the subjecthaving suffered from mTBI, wherein decreasing SNTF levels indicates asubsequent decreased risk of brain damage, long-term functionaldisability, or long-term neurological dysfunction.